Cell architecture of beech (<i>Fagus grandifolia</i>) leaves.

Photomicrographs of transverse sections of control and symptomatic beech leaf disease (BLD) leaves collected in early autumn. Sections were stained with a mixture of hematoxylin and eosin. (A) Representative control beech leaf. (B-C) Section of a mature control beech leaf showing differentiated cell layers as indicated in figure C. (D) Representative symptomatic BLD leaf. Dotted white, blue and orange squares represent sections of (G) asymptomatic, (H) green and (E-F) yellow banded areas of the same leaf used for histological analyses. (E-F) Sections of yellow symptomatic BLD areas showing extensive leaf thickness as a consequence of cell proliferation and cell hypertrophy. Nematode sections are stained (dark purple). (G) Section of a non-symptomatic area of a BLD leaf, which presents cell architecture similar to that of control leaves (B). (H) Section of a green interveinal area of a BLD leaf without nematodes. Note the additional cell layers in comparison to the control leaves (C). (I) Leaf thickness was measured from photomicrographs of control, asymptomatic (BLD_a) and symptomatic BLD areas without (BLD -n) and with (BLD +n) nematodes. For each condition a minimum of 50 measurements were made. (J-L) Cell surface (μm2) was measured for the upper epidermis (J), lower epidermis (K) and spongy mesophyll cells (L). Measures were made for control (n = 80 cells), asymptomatic (BLD_a, n = 100 cells) and symptomatic BLD areas without (BLD -n, n = 100 cells) and with (BLD +n, n = 100 cells) nematodes. Thick lines in the boxplot represent medians. Different letters denote statistical differences between cell sizes (P < 0.05, ANOVA with Tukey-Kramer test). Scale bars: 100 μm (A, B); 50 μm (C-H).</p

Measurement and weight of 50 buds collected from symptomatic beech leaf diseased trees in late February of 2023.

Measurement and weight of 50 buds collected from symptomatic beech leaf diseased trees in late February of 2023.</p

Measurement and weight of 50 poor developing beech buds collected in early spring of 2023.

Measurement and weight of 50 poor developing beech buds collected in early spring of 2023.</p

Sections of young developing leaves within nematode-infected (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) beech buds (<i>Fagus grandifolia</i>) with altered interveinal leaf cell proliferation.

Morphological cell rearrangements of interveinal leaf cross-sections. (A) Representative section of control leaf interveinal areas with the typical six layers of cells. (B-D) Three interveinal areas of the same leaf infected by nematodes showing a variable and prominent increase in the number of cells layers. Outline drawings of some representative interveinal areas highlighting this upsurge in cell division associated with nematodes (B’-C’) compared to the control (A’). (E) Veinal areas of the same nematode-infected leaf displayed different levels of ectopic cell proliferation. (F) Differential cell proliferation within the same interveinal leaf area dissected from a nematode-infected bud. Scale bars: 20 μm.</p

Nematode (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) assessment of leaves and beech (<i>Fagus grandifolia</i>) buds in early spring.

(A) Representative branch showing sets of asymptomatic and symptomatic beech leaf disease (BLD) leaves, and poorly developing buds. (B-C) Detection of nematodes in symptomatic BLD leaves using acid fuchsin staining (B). Nematodes (arrows) were found migrating on the abaxial side of the leaf and penetrating the leaf tissues (C-C’). (D) Representative symptomatic BLD leaves dissected from poorly developing buds. (E) Percentage of asymptomatic and symptomatic BLD leaves associated with 50 poor-developing buds rated according to the phenotype scale presented in Fig 1. (F) Active nematodes and eggs extracted from a single, poorly developing bud. (G) Numbers of nematodes extracted from 50 poorly developing buds. Buds were classified from 0 to 4 based on their bud scale phenotype (see Fig 1). Scale bars: 5 mm (B); 1 mm (C, D, F); 500 μm (C´).</p

Nematode (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) and leaf primordia development in dormant buds of beech (<i>Fagus grandifolia</i>).

Fifty buds were randomly collected from symptomatic beech leaf diseased (BLD) trees in late February. (A) Representative bud scale cells of control and nematode-infected buds. Cell hypertrophy is associated with the presence of nematodes. (B) Representative leaves collected from control and nematode-infected buds. Buds infected with a high population of nematodes caused highly symptomatic and damaged leaves. (C) Number of nematodes quantified for 50 randomly collected buds. Both active and dead nematodes were quantified and represented by different colors in a bar graph. Buds were phenotyped according to the previously established groups (see Fig 1). (D) Percentage of asymptomatic and symptomatic BLD leaves associated with 50 dormant buds distributed by their phenotype. Scale bars: 50 μm (A); 1 mm (B).</p

Examples of enlarged cell associated with nematode-infected (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) bud scales.

(A-C) Nematode-infected bud scales showing different levels of cell hypertrophy within scales of the same bud (arrows point out nematodes). (D) Clusters of eggs and motile stages found in-between the bud scales. Scale bars: 50 μm. (PDF)</p

Detection of <i>Litylenchus crenatae</i> subsp. <i>mccannii</i> collected from the bark of beech twigs.

(A-B) Nematodes collected from twigs. (C) Amplification curves obtained from four sets of nematodes collected from independent beech twigs (red). Nematodes collected from buds were used as positive control (blue). Water was used as negative control (orange). Nematode detection was conducted using a fatty acid- and retinol-binding gene (FAR) of L. crenatae subsp. mccannii. Scale bars: 100 μm. (PDF)</p

Nematode-infected (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) bud scale cells showed variable levels of hypertrophy.

(A-C) Photomicrographs of control bud scales showing flat, rectangular-shaped cells. (D-F) Representative infected bud scale cells. Infected cells presented several levels of hypertrophy and a more disorganized distribution of the cells (F). (G) Different nematode stages (arrows) associated with abnormally large scale cells. (H) Example of nematodes (arrows) probing large scale cells. (I) Large numbers of eggs were often found in nematode infected scales. Scale bars = 50 μm.</p

Scanning electron photomicrographs of transverse sections of control and symptomatic beech (<i>Fagus grandifolia</i>) leaves.

(A) Transverse section of a control leaf showing the typical composition of six layers of cells. (B) Transverse section of a crinkled leaf (early spring) showing an abnormal number of cell layers and very compact intercellular spaces in the spongy mesophyll. (C) Representative transverse section of a symptomatic leaf showing asymptomatic (left) and BLD symptomatic (right) areas. (D-E) Symptomatic BLD section with numerous nematodes (arrows) within the spongy mesophyll. Scale bars: 20 μm (A, B); 100 μm (C-E).</p